Urbieta et al. BMC Genomics (2017) 18:445 DOI 10.1186/s12864-017-3828-x RESEARCH ARTICLE Open Access Genome analysis of the thermoacidophilic archaeon Acidianus copahuensis focusing on the metabolisms associated to biomining activities María Sofía Urbieta1,3*†, Nicolás Rascovan2†, Martín P. Vázquez2 and Edgardo Donati1 Abstract Background: Several archaeal species from the order Sulfolobales are interesting from the biotechnological point of view due to their biomining capacities. Within this group, the genus Acidianus contains four biomining species (from ten known Acidianus species), but none of these have their genome sequenced. To get insights into the genetic potential and metabolic pathways involved in the biomining activity of this group, we sequenced the genome of Acidianus copahuensis ALE1 strain, a novel thermoacidophilic crenarchaeon (optimum growth: 75 °C, pH 3) isolated from the volcanic geothermal area of Copahue at Neuquén province in Argentina. Previous experimental characterization of A. copahuensis revealed a high biomining potential, exhibited as high oxidation activity of sulfur and sulfur compounds, ferrous iron and sulfide minerals (e.g.: pyrite). This strain is also autotrophic and tolerant to heavy metals, thus, it can grow under adverse conditions for most forms of life with a low nutrient demand, conditions that are commonly found in mining environments. Results: In this work we analyzed the genome of Acidianus copahuensis and describe the genetic pathways involved in biomining processes. We identified the enzymes that are most likely involved in growth on sulfur and ferrous iron oxidation as well as those involved in autotrophic carbon fixation. We also found that A. copahuensis genome gathers different features that are only present in particular lineages or species from the order Sulfolobales, some of which are involved in biomining. We found that although most of its genes (81%) were found in at least one other Sulfolobales species, it is not specifically closer to any particular species (60–70% of proteins shared with each of them). Although almost one fifth of A. copahuensis proteins are not found in any other Sulfolobales species, most of them corresponded to hypothetical proteins from uncharacterized metabolisms. Conclusion: In this work we identified the genes responsible for the biomining metabolisms that we have previously observed experimentally. We provide a landscape of the metabolic potentials of this strain in the context of Sulfolobales and propose various pathways and cellular processes not yet fully understood that can use A. copahuensis as an experimental model to further understand the fascinating biology of thermoacidophilic biomining archaea. Keywords: Acidianus copahuensis, Thermoacidophilic archaea, Biomining genes * Correspondence: [email protected] †Equal contributors 1CINDEFI (CCT La Plata-CONICET, UNLP), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Calle 47 y 115, 1900 La Plata, Argentina 3Calle 50, entre 115 y 116, N° 227, La Plata, Buenos Aires, Argentina Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Urbieta et al. BMC Genomics (2017) 18:445 Page 2 of 14 Background from this group, together with further experimental Biomining comprises technological processes (bioleach- characterization will undoubtedly bring new insights ing and biooxidation) that use microorganisms, usually into the biology of these organisms. bacteria and archaea, to enhance the recovery of metals A. copahuensis is a novel thermoacidophilic archaeon from insoluble ores mostly composed of metal sulfides. from the domain Crenarchaeota and the order Sulfolo- For the solubilization of sulfides, two conditions are re- bales, isolated by our group from the acidic Copahue quired: an oxidizing agent and an acidic medium to geothermal area in the Northwest corner of the Cordil- maintain the removed metal cations in solution. Both lera de los Andes in Neuquén province (Argentina). It conditions can be met by acidophilic iron- and sulfur has shown a great physiological flexibility by growing in oxidizing microorganisms; they can oxidize ferrous iron a temperature range of 55 °C to 80 °C and pH range to ferric iron (a powerful oxidizing agent), and also from 1 to 5, with optimum conditions at 75 °C and oxidize metal sulfides and sulfur compounds to sulfuric pH 3, respectively [11]. Its metabolic features make it an acid [1]. Most of the commercial applications are imple- excellent candidate for biomining of sulfide minerals as mented at moderate temperatures, below 50 °C, mainly it is able to oxidize diverse sulfur compounds (sulfur, tet- because the firsts and best characterized bioleaching rathionate and metal sulfides such as pyrite and chalco- species are mesophiles. However, a higher operational pyrite), and ferrous irons, either heterotrophically or temperature would be significantly beneficial as it would autotrophically, being the latter a valuable attribute in allow a reduction in the energy used for cooling the sys- mining environments, where organic carbon is often tem (sulfur oxidation reactions are exothermic, causing limited. We have experimentally shown that A. copa- a serious increase in temperature in bioreactors and in- huensis is able to recover a 100% of copper in the bio- side the heaps) and would decrease the inconveniencies leaching of a chalcopyrite concentrate [7]. In addition, associated to mineral surface passivation [2]. Probably A. copahuensis can grow in anaerobic conditions using themostrelevantexampleisthe case of chalcopyrite sulfur or hydrogen as electron donors and ferric iron or (CuFeS2), a mineral species that accounts for approxi- sulfur as electron acceptors, an essential adaptation for mately 70% of the world’s copper reserves [3] and is the anoxic conditions found below heaps surface [12]. In highly recalcitrant to chemical or mesophilic bio- the present work we characterized the genome of this logical leaching [4]. In the past few years, several remarkable biomining candidate and the genes associ- studies have shown that thermoacidophilic archaea ated to its capabilities, such as the oxidation and reduc- are able to obtain faster solubilization rates and tion of sulfur and iron compounds, electron transport, higher copper recovery yields than most used meso- carbon fixation, tolerance and resistance to heavy metals philic bioleaching bacteria [5–7]. and metalloids. We also performed a comprehensive The physiological, biochemical and genetic comparison of A. copahuensis genome with all other characterization of thermoacidophilic archaea, especially available genomes from the order Sulfolobales and found the features related to biomining, became a topic of that it groups different features that are only found interest some years ago and some advances were made within specific genera of this order. on elucidating the genes and metabolic pathways in- volved in the oxidation of sulfur compounds and ferrous Results and discussion iron. However, none of them are yet completely under- Acidianus copahuensis within the order Sulfolobales stood. The key enzymes for sulfur oxidation in thermoa- A total of 2559 genes were predicted in Acidianus copa- cidophilic archaea, the sulfur oxygenase reductase (SOR) huensis ALE1 strain (DSM 29038) genome using the and the thiosulfate quinone oxidoreductase (TQO), have RAST annotation server. The comparison to all other been characterized in Acidianus ambivalens [8]. Regard- available genomes of the order Sulfolobales at the whole ing iron oxidation, a cluster of genes up-regulated when genome level using an in silico DDH method showed cultures were grown in ferrous iron was identified in only a 30% similarity to the closest genome and only Sulfolobus metallicus; thus this cluster, named fox,was 15% to Acidianus hospitalis, the other sequenced species directly linked to ferrous iron metabolism [9]. These within the genus Acidianus (Table 1). genes are not present in other Sulfolobus species that do According to a network analysis comparing all pro- not oxidize iron. Some other biomining related features teins from Sulfolobales genomes, Acidianus copahuensis were also identified in the genome of Metallosphaera is not closer to any particular genus among Sulfolobales sedula, such as carbon fixation, metal resistance, and ad- (Fig. 1a). It shares around two thirds (min: 50%, max: hesion mechanisms [10]. Despite the light that these 68%, avg.: 64%) of its proteins with each of the other works shed into the unexplored bioleaching mechanisms Sulfolobales species (Additional file 1: Figure S1) and of thermophilic archaea, many aspects of their metabo- 39% of them (1003) are core proteins present in all ge- lisms remain still unclear. The analysis of new genomes nomes (Fig. 1b, Additional file 2: Table S1). In fact, the Urbieta et al. BMC Genomics (2017) 18:445 Page 3 of 14 Table 1 Digital DDH estimation